增强型绿色荧光蛋白在集胞藻6803中的表达

利用聚球藻7942热休克基因groESL的启动子和报告基因egfp,构建了表达载体pUC-Tegfp并转化集胞藻6803,并通过所制备抗体对转基因藻进行蛋白免疫印迹检测.结果发现,在转基因藻株T-egfp的细胞粗提液中含有能与eGFP抗体特异结合的蛋白质,表明外源增强型绿色荧光蛋白基因(egfp)在集胞藻6803中成功表达.     

钝顶螺旋藻表达载体p215 t-spp的构建及转化

用合适的限制性内切酶消化载体p215t,胶回收获得含有gfp基因及amp基因大片段.设计引物,采用PCR扩增无菌钝顶螺旋藻A9藻株的强启动子片段;在T4连接酶的作用下进行体外连接重组,构建了带有螺旋藻启动子和gfp报告基因的新型表达载体p215t-spp.将该载体转入钝顶螺旋藻,利用报告基因的表达,在荧光显微镜下观察并记录转化藻细胞,p215t-spp质粒显著提高转化率.研究了不同PEG浓度、转化时间及冰浴处理对转化率的影响.采用1%PEG能有效促进细胞吸收DNA,获得10.5‰的转化率.实验初步证明,带有螺旋藻启动子的报告基因gfp能够在钝顶螺旋藻中顺利表达.     

外源报告基因EGFP在盐藻中实现瞬时表达

探索杜氏盐藻 (Dunaliellasalina)的转基因方法和筛选方法 .利用烟草花叶病毒启动子 (CaMV35S)、衣藻叶绿体atpA启动子与来源于水母的加强型绿色荧光蛋白报告基因 (EGFP)构建表达载体pART7GFP和pUCGFP ,转化盐藻 .EGFP在CaMV 35S启动下表达出绿色荧光蛋白 ,在荧光显微镜下看到发绿色荧光的转基因盐藻 .根据荧光数目进行统计 ,转化效率高于 5 % .衣藻来源的启动子atpA在盐藻中未能启动EGFP的表达 .用直径 1μm的金粉颗粒和 0 6 μm的金粉进行基因枪法转化 ,1μm的金粉颗粒成功将外源基因导入盐藻 ,用 0 6 μm的金粉配合多种技术参数也没有将外源基因导入盐藻 .EGFP可以用作盐藻遗传转化的报告基因使单细胞真核生物盐藻可以利用流式细胞术 (FACS)等技术进行筛选 ,从而避开平板筛选转基因盐藻的限制 ,并使转基因盐藻实现无抗生素筛选成为可能     

高中生物学遗传与变异实验课教学案例开发

通过接合作用,大肠杆菌携带的质粒载体pFAJ1819::mTn5gusA -pgfp21转入目标受体菌催娩克氏菌;载体上的转座子mTn5gusA -pgfp21携带的葡萄糖苷酸酶基因(gusA,无启动子)、卡那霉素抗性基因(nptⅡ,有启动子)和2个绿色荧光蛋白基因(gfp,有启动子)插入到催娩克氏菌的基因组DNA中;借助绿色荧光蛋白、gusA及卡那霉素抗性基因的表达,形成的表型直观变化展示细菌的遗传与变异现象,可以作为高中生物学实验的教学案例.     

绿色荧光蛋白gfp基因在钝顶螺旋藻(Spirulina platensis)A9藻株中的表达

将钝顶螺旋藻(Spirulina platensis)A9藻株在24℃培养,经2 mmol/L的EDTA预处理24 h;采用功率300 W的超声波处理70 s获得单细胞样品,以本实验室构建的携带gfp基因的质粒p215t转化A9藻株单细胞藻液,利用Amp作为选择标记,使单细胞在平板上再生长出单藻落,获得17株具有Amp抗性的转化藻株,转化率3.73‰。在390 nm紫光激发下,生长30天的转化藻丝体发出稳定绿色荧光;培养45天后具有绿色荧光的藻丝出现断裂、具有荧光藻丝长度缩短的现象。实验结果初步表明:报告基因gfp在螺旋藻中得到稳定有效的表达,可以采用单细胞再生形成单藻落技术进行螺旋藻的基因克隆。     

根癌农杆菌介导的灰葡萄孢菌遗传转化研究

以pCAMBIA1300-N载体为骨架, 成功构建了以绿色荧光蛋白(gfp)为报告基因, 潮霉素(hph)为抗性筛选标记的载体pKPG, 并利用根癌农杆菌介导转化系统, 成功获得了能表达绿色荧光蛋白的重组灰葡萄孢菌。通过PCR检测转化子的绿色荧光蛋白基因和潮霉素抗性表达框, 观察菌丝和分生孢子的荧光表型, 以及gfp基因的Southern杂交验证, 结果表明：被测转化子基因组中均成功整合了目的基因片段。     

集胞藻PCC6803中基因slr1761突变体的构建

PCR扩增了集胞藻PCC6803的slr1761基因,进一步以PGEM-T为载体将其克隆到大肠杆菌中,构建了P1761质粒。通过DNA体外重组,以卡那霉素抗性基因插入目的基因片段,构建了既含目的基因上游及下游序列、又携带选择性标记卡那霉素抗性的PK1761质粒。该质粒转化野生型集胞藻PCC6803细胞,利用同源重组原理获得了能在含卡那霉素的培养基上正常生长的基因敲除突变株。对该突变株基因组DNA进行PCR扩增,验证了其基因结构的正确性。     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

小鼠脂联素与绿色荧光蛋白融合基因表达载体的构建及在COS-7细胞中的表达

以绿色荧光蛋白(GFP)基因(gfp)为报告基因,构建小鼠脂联素(mADPN)基因(mAd)与gfp的融合基因mAd/gfp表达载体pCI-neo-apoEHCR-hAATp-mAd-gfp,脂质体法转染体外培养的COS-7细胞,荧光显微镜观察GFP在细胞中的表达可间接反映mADPN的表达,并通过RT-PCR在核酸水平进一步确证mAd的表达.荧光显微镜观察及RT-PCR结果均证明mADPN在COS-7细胞中获得了高效表达,表明mADPN重组表达载体pCI-neo-apoEHCR-hAATp-mAd可以在真核细胞COS-7中高效表达mADPN,为进一步探讨mAd在小鼠体内的表达提供了可行性依据.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp , produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfp s as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.     

Identification of macrophage induced genes of Corynebacterium pseudotuberculosis by differential fluorescence induction

Corynebacterium pseudotuberculosis is the etiological agent of the sheep disease caseous lymphadenitis. We have developed a promoter reporter system for this organism based on expression of the green fluorescent protein (gfp) gene from Aequorea victoria. A promoterless vector, pSM20, containing the gfp gene was constructed, and promoters were inserted upstream of the gfp gene. Upon transformation into C. pseudotuberculosis, fluorescence could be visualised by fluorescence microscopy, and relative promoter strength measured by flow cytometry. The usefulness of this system for measuring changes in gene expression was demonstrated by measuring fluorescence levels of heat shocked C. pseudotuberculosis carrying a dnaK promoter construct. Replication of C. pseudotuberculosis within J774 macrophages could be monitored by fluorescence microscopy. The establishment of the system allowed the use of differential fluorescence to identify genes that showed up-regulation following macrophage infection. Genes coding for a non-ribosomal peptide synthetase and the beta chain of a propionyl CoA carboxylase were identified as possessing promoters that demonstrated enhanced activity following macrophage infection by C. pseudotuberculosis.     

控制基因盐诱导且根系优势表达的小麦启动子Tasipro3克隆及功能分析

在土壤盐胁迫下,小麦根系吸收水分和营养物质的功能受到抑制,从而影响植株的经济产量。因而,开展小麦耐盐育种,提高根系耐盐性是重要途径之一。使耐盐基因在根系中优势表达,并且在盐胁迫下增强表达,将显著提高根系耐盐性。而克隆和鉴定具有双重控制功能的启动子,是实现耐盐基因精准调控的基础。鉴于此,本研究利用Genevestigator在线生物信息学分析软件,筛选到425个盐诱导根系优势表达的探针,并从中选出2个候选探针,用于启动子验证。以1周龄小麦品种中国春的幼苗为材料,将其根系置于200 mmol/L的NaCl溶液中,分别于0 h、0.5 h、1 h、2 h、4 h和8 h进行根系取样,用于表达模式分析。结果表明,Ta.5463.1.A1_at探针的基因表达模式更符合生物信息学预测的结果,受盐胁迫诱导表达显著上调,且基因优势表达于根系。为进一步验证相应基因启动子的功能,对此探针对应的启动子区进行了克隆,并连接到启动子验证载体中,遗传转化获得转基因拟南芥植株。盐诱导后GUS染色的实验结果表明,该启动子使GUS报告基因在盐处理下表达量显著提高,且主要在根系表达。本研究成功克隆了耐盐遗传改良专用启动子,为小麦分子抗逆育种提供了优异资源。     

Noninvasive fluorescent screening of microinjected bovine embryos to predict transgene integration

Most transgenic domestic animals are generated by direct microinjection of DNA fragments into zygote pronuclei. It has generally been assumed that the majority of integration events should occur prior to the first round of chromosomal DNA replication. The aim of this study was to investigate the expression of GFP in bovine preimplantation embryos by using a gfp reporter gene consisting of chicken beta-actin promoter, the CMV-IE enhancer, gfp cDNA (EGFP) (732 bp) and rabbit beta-globin polyadenylation sequences. In five experiments 302 bovine zygotes were injected while 75 served as a control. The fluorescence intensity was detected at 72 and 168 h following fertilization in bovine embryos injected with 3 ng/microl in experiments 1-3, and injected with 5 ng/microl in experiments 4-5. Eight embryos were considered as expressing green fluorescence protein; 2 of them were 100% fluorescent after microinjection of a higher dose of the DNA; one was 75%, two--50%, and three 25% transgenic. The mosaicism was assumed to be at 75%. The results indicated that the fluorescence could be analyzed at any time of bovine embryo development. It was therefore concluded, that chicken beta-actin promoter together with the CMV-IE enhancer would confer a strong expression of the gfp reporter gene in preimplantation bovine embryos. Therefore, using GFP that could be simply detected in live bovine (transgenic) embryos would be very promising in establishing transgenic lines of domestic animals producing in their fluids human therapeutic proteins.